JP5414830B2 - Sphingomyelin-containing medicine - Google Patents

Description

  The present invention relates to a medicament containing sphingomyelin and having a novel use. More specifically, the present invention comprises sphingomyelin, a sialomucin secretion promoter, a sickness prevention agent, an antiallergic agent, an antioxidant, an infection defense agent, a hair nourishing agent, a demyelinating disease therapeutic agent, an antipigmentation agent, Or it is related with the pharmaceutical which is an anti-inflammatory agent, the food-drinks or feed which mix | blended these agents.

Sphingomyelin is a kind of phospholipid and is abundant in milk, and accounts for about 30% of phospholipid in milk. Sphingomyelin has a structure in which phosphocholine is bound to a ceramide skeleton composed of sphingosine and a fatty acid, and is known to exist in the brain and nerve tissues. It is also reported that it is slightly contained in foods such as egg yolk.
It is known that sphingomyelin influences cell proliferation and differentiation in vivo via an information transmission system. Furthermore, although it is known that it has an action to improve the digestion and absorption function of lipids accompanying aging (Patent Document 1), little is known about other actions. Therefore, development of medicines, foods and drinks and feeds containing sphingomyelin as an active ingredient is expected.

JP-A-11-269074

  An object of the present invention is to find a novel pharmaceutical use of sphingomyelin, and to provide a pharmaceutical effective as a preventive or therapeutic agent for various diseases, and a food and drink and a feed containing these agents.

  In order to solve the problem, the present inventors conducted various studies on the pharmacological effects of sphingomyelin. As a new use, sphingomyelin is a sialomucin secretion promoting effect, sickness prevention effect, antiallergic effect, antioxidant. The present invention was completed by finding that it has an effect, an infection-protecting effect, a hair-restoring effect, a demyelinating disease treatment effect, an anti-pigmentation effect or an anti-inflammatory effect.

That is, the present invention contains a sphingomyelin as an active ingredient, and is a pharmaceutical which is one of the following agents: 1) anti-drunk agent, 2) anti-allergic agent, 3) antioxidant, 4) anti-infective agent, 5 A hair nourishing agent, 6) a demyelinating agent, 7) an anti-pigmentation agent, or 8) an anti-inflammatory agent.
The present invention is also the above-mentioned pharmaceutical, wherein sphingomyelin is derived from milk.

  According to the present invention, sphingomyelin is converted into 1) an agent for preventing sickness, 2) an antiallergic agent, 3) an antioxidant, 4) an anti-infective agent, 5) a hair nourishing agent, 6) a therapeutic agent for demyelinating disease, and 7) an anti-pigment. It can be used as a deposition agent or 8) an anti-inflammatory agent.

It is a graph which shows the result of the tight bond formation test of this invention.

  The sphingomyelin that can be used in the present invention is not particularly limited, and is chemically synthesized, naturally derived, for example, derived from milk such as milk or goat milk, or derived from egg yolk such as chicken egg. Although the thing is mentioned, the thing derived from milk is more preferable. Among the milk, the sphingomyelin raw material derived from milk is particularly preferable because the sphingomyelin content is as high as 25% or more, and inexpensive ones are commercially available.

The sphingomyelin may be purified and purified, or may be used in the form of a phospholipid containing sphingomyelin.
Regarding sphingomyelin and sphingomyelin-containing phospholipids, for example, milk-derived sphingos obtained by a method of extracting dairy products such as milk and whey protein concentrate (WPC) with ether or acetone (JP-A-3-47192). Myelin-containing phospholipids (containing about 28% by weight of sphingomyelin in phospholipids) can be used. Further, an aqueous fraction containing butter curd and butter serum obtained by heating and melting butter can be used as sphingomyelin-containing phospholipid (containing about 9% by weight of sphingomyelin in phospholipid). Furthermore, the milk fat globule membrane fraction contained in buttermilk or buttersarum can be used as sphingomyelin-containing phospholipids (containing about 9% by weight of sphingomyelin in phospholipids). And, using sphingomyelin with increased purity by purifying these sphingomyelin-containing phospholipids by methods such as dialysis, ammonium sulfate fractionation, gel filtration, isoelectric precipitation, ion exchange chromatography, solvent fractionation, etc. Also good.

The medicament of the present invention can be used as a preparation having various dosage forms, and the dosage form is not particularly limited. Therefore, sphingomyelin and / or phospholipids containing sphingomyelin can be blended in various dosage forms such as tablets, capsules, powders, and liquids.
In addition, the type of food and drink of the present invention is not particularly limited, and food and drink such as milk, processed milk, milk drink, yogurt, soft drink, coffee drink, juice, jelly, wafer, biscuit, bread, noodle, sausage and the like It can mix | blend with a nutritional supplement and the composition for a nutritional supplement. Moreover, the kind of feed of this invention is not specifically limited.
In addition, the pharmaceutical of this invention, food-drinks, and feed can be manufactured by a conventional method except containing sphingomyelin.

  In this invention, in order to exhibit each pharmacological effect, although it changes with uses, as a sphingomyelin, the compounding quantity to a pharmaceutical, food-drinks, and feed so that about 0.1-100 mg can generally be ingested per day. Etc. may be adjusted.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples. “%” In Examples and Test Examples means “% by weight” unless otherwise specified.
Example 1
A reaction solution obtained by allowing protease to act on a 10% aqueous solution of whey protein concentrate (WPC) was extracted with a chloroform-methanol (2: 1) solution, concentrated, and further extracted with acetone to obtain a complex lipid fraction. Got. Next, this complex lipid fraction was step-extracted with a chloroform-methanol solution by Florisil column chromatography to obtain a phospholipid fraction. This phospholipid fraction was subjected to phase extraction with a chloroform-methanol solution by silica gel chromatography, and the fraction obtained was freeze-dried to obtain a sphingomyelin raw material. This sphingomyelin raw material was processed by thin layer chromatography, then developed with a Dittmer reagent, and the sphingomyelin content was measured by densitometry and found to be 95.2%. This sphingomyelin raw material can be used as it is as the agent of the present invention.

(Test Example 1)
The effect of sphingomyelin on the promotion of sialomucin secretion was tested using the method of “Test Example 1” in Japanese Patent Application Laid-Open No. 2001-206848.
That is, the control group (Control) is a standard diet of AIN-93G, and the sphingomyelin administration group (SPM) is a portion of the sucrose of the standard diet as a sphingomyelin raw material described in Example 1 of this specification. Further, the sialyl lactose-administered group (SL) was administered with a feed in which 1% of the sucrose in the standard diet was replaced with sialyl lactose.
Seven-week-old SD male rats (manufactured by Charles River Japan) were bred under conditions of 60% humidity, room temperature 24 ° C., and light-dark control for 12 hours. All rats were preliminarily raised on a standard diet for 1 week, divided into 3 groups of 12 animals per group, and allowed to freely feed each experimental diet for 1 week. Rat saliva was collected on the 7th day after administration of the experimental diet, and the sialomucin content in the saliva was measured by high performance liquid chromatography. Table 1 shows the measurement results of the sialomucin content in the saliva of each experimental group. As is clear from the results shown in Table 1, the sphingomyelin administration group had a markedly increased sialomucin content compared to the control group, and also increased compared to the sialyl lactose administration group.

(Test Example 2)
The effect of sphingomyelin on promoting the secretion of sialomucin was tested using the method for testing the cholera toxin binding inhibitory activity of “Test Example 2” in Japanese Patent Application Laid-Open No. 2001-206848.
The binding inhibition activity of cholera toxin was examined using the saliva of each experimental group of Test Example 1 of this specification. 200 μl of a 0.1% ganglioside GM1-containing ethanol solution (w / v) was added to a 96-well ELISA test plate, and then air-dried to adsorb ganglioside GM1. Saliva from each experimental group was diluted 10-fold with PBS containing 1% bovine serum albumin (BSA), and then biotin-conjugated cholera toxin was added and allowed to react for 1 hour. 100 μl of the reaction solution was added to the above ELISA test plate and allowed to stand for 30 minutes, and then the supernatant was removed. The ELISA test plate was washed several times with PBS containing 0.05% Tween 20, biotin-binding β-galactosidase was added and allowed to stand for a certain time, and then the supernatant was removed. In addition, the ELISA test plate was washed several times with PBS containing 0.05% Tween 20, and 4-methylumbelliferyl galactose was added and reacted for 30 minutes. Measured with a fluorometer (excitation wavelength 360 nm, measurement wavelength 460 nm). And the rejection rate was computed from following Formula.
Blocking rate (%) = {1- (A / B)} × 100
A: Fluorescence intensity of sphingomyelin administration group (SPM) and sialyl lactose administration group (SL) B: Fluorescence intensity of control group (Control) The results are shown in Table 2. As is clear from the results in Table 2, it was found that the binding inhibition activity of cholera toxin in the sphingomyelin administration group was very high compared to the control group. Furthermore, it was found that the binding inhibition activity of cholera toxin in the sphingomyelin administration group was higher than that in the sialyl lactose administration group. Therefore, it was found that oral ingestion of sphingomyelin increases the content of sialomucin in saliva and, as a result, enhances the ability to neutralize toxins.

In addition, the measuring method of the sialomucin content in saliva was as follows.
(1) Collection of saliva Rats fasted for 2 hours or more were intramuscularly injected with 0.2 ml of Nembutal solution, and after anesthesia, a pilocarbine hydrochloride solution, which is a salivary secretion promoter, was intramuscularly injected. After 3 minutes, saliva secreted under the rat tongue was collected into a microtube by an autopipette, and this operation was performed for exactly 9 minutes. After completion of saliva collection, 0.1 ml of 0.1% atropine sulfate, which is a saliva secretion inhibitor, was injected to terminate saliva collection.
(2) Collection of sialomucin fraction The collected saliva was rapidly refrigerated to 0 ° C. or lower, and then treated with a centrifuge cooled to 4 ° C. (11,000 rpm, 60 minutes) to obtain a supernatant. The supernatant was dialyzed against physiological saline with a microdialysis tube having a molecular weight fraction of 100,000 for 3 days, and the contents were collected as a sialomucin fraction in saliva.
(3) Quantification of sialomucin content The sialomucin content contained in the siaromutin fraction was quantified with a sialic acid fluorescent labeling kit (manufactured by Takara). A certain amount of sialomucin fraction was collected in a test tube and dried under reduced pressure using a rotary evaporator, and then 2N-acetic acid was added and hydrolyzed at 80 ° C. for 3 hours. The released N-acetyl sialic acid and O-acetylated sialic acid were quantified by high performance liquid chromatography after adding a DMB reagent as a fluorescent labeling agent and reacting at 55 ° C. for 2.5 hours.

(Test Example 3)
The effect of sphingomyelin on the prevention of sickness was tested by the method of “Example 1” of Japanese Patent Application Laid-Open No. 2001-199880.
Wistar male rats were used at a body weight of 110-120 g after 1 week of preliminary breeding. Fasted overnight before the experiment, and fasted and dehydrated during the experiment. Rats were divided into an alcohol-only administration group (control group) and a sphingomyelin administration group, and 5 rats per group were used.
The 40% (v / v)% aqueous ethyl alcohol solution was 10 ml / kg for the alcohol alone administration group (control group), and the 40% (v / v)% ethyl alcohol aqueous solution was 10 ml / kg for the sphingomyelin administration group. The sphingomyelin raw material 2 mg / kg of Example 1 was orally administered. After administration, observations were made until the symptoms of sickness recovered.
As a result, in the alcohol alone administration group (control group), it took 5 hours for the sickness symptoms to recover, and when administered, the symptoms were prone, staggered walking, and decreased grip strength, whereas the sphingomyelin administration group The symptoms of drunkenness recovered within 1 hour, and the symptoms were slightly staggered when administered. From this, it was confirmed that sphingomyelin has a remarkable effect of preventing sickness symptoms during drinking.

(Test Example 4: Tight bond formation test)
The antiallergic action of sphingomyelin was tested by the method of “Test Example 1” of JP-A-8-109133.
To the serum-free medium Cosmedium001 (manufactured by Cosmo Bio), ganglioside GM3 or sphingo so that the concentration of ganglioside GM3 (manufactured by Sigma) or the sphingomyelin raw material of Example 1 of the present specification is 2 μg / ml, respectively. Each myelin was added. Millicell-CM (manufactured by Millipore; pore size 0.4 μm; 0.6 cm 2) was placed on a 24-well microtiter plate, and the membrane surface was treated with collagen (manufactured by Koken), and then a human colon adenocarcinoma cell line Caco. -2 was cultured, and the following points were compared with the case of culturing in 10% FCS-added medium (10% FCS) and Cosmedium 001 (no addition) to which nothing was added. That is, the electrical resistance (R) value inside and outside of Millicell-CM was measured on days 1, 4 and 7 of culture using an electrical resistance measuring device Millicell-ERS (manufactured by Millipore). The results are shown in FIG. As is clear from the results shown in FIG. 1, in the sphingomyelin supplemented medium (SPM), the R value increased as in the ganglioside GM3 supplemented medium (GM3) and the 10% FCS supplemented medium. From this, it is considered that in the sphingomyelin and GM3-added medium, the tight binding of Caco-2 progressed and the gap between cells decreased. In contrast, the R value did not increase in the additive-free medium. Therefore, it was found that sphingomyelin has an effect of tightly binding intestinal mucosal cells and has an antiallergic action by preventing the entry of allergens into the body.

(Test Example 5: Secretory IgA production promotion test)
The antiallergic action of sphingomyelin was tested by the method of “Test Example 2” of JP-A-8-109133.
5 ml of aseptically collected human breast milk was diluted 2-fold with a 10 mM phosphate buffer solution (PBS; Phosphate buffered saline, pH 7.2) containing 150 mM NaCl, and then the separated solution [33.4% Conray 400 (Daiichi Pharmaceutical Co., Ltd.). Solution prepared by mixing 5:12 with 9% Ficoll (manufactured by Pharmacia)] The test tube containing 5 ml was layered. After centrifugation at 400 × G for 30 minutes, the intermediate layer in which lymphocytes were collected was collected with a Pasteur pipette. Lymphocytes were dispersed in 10 ml of PBS and washed, and then centrifuged at 150 × G for 10 minutes. After this washing operation was repeated three times, 12 ml of RPMI-1640 medium containing insulin (10 μg / ml) and transferrin (5 μg / ml) was added, and 3 ml each was dispensed into three petri dishes (A; B; C). . In petri dish A, 0.3 ml of fetal calf serum (FCS) was added, in petri dish B, 3 μg of the sphingomyelin raw material of Example 1 of this specification was added, and nothing was added to petri dish C.
Seven days later, the IgA content in the culture broth was 3.89 μg / ml for Petri dish A, 3.11 μg / ml for Petri dish B, and less than 0.1 μg / ml for Petri dish C. In Petri dishes A and B, IgA production was high, but in Petri dish C, IgA was hardly produced. From this result, it was found that sphingomyelin has an effect of increasing IgA production ability of lymphocytes and has an antiallergic action.

(Test Example 6: Allergen invasion inhibitory effect test)
The antiallergic action of sphingomyelin was tested by the method of “Test Example 3” in JP-A-8-109133.
Infant Wistar rats (14 days old, body weight around 20g, 8 animals, Charles River Japan) were divided into a control group and a sphingomyelin administration group (SPM administration group), both of which approximated to rat milk Breeding with artificial milk. On the 14th to 20th days, the SPM administration group was orally administered daily with 50 μl of a sphingomyelin solution (1 mg / ml) prepared from the sphingomyelin raw material of Example 1 of this specification using a micropipette. On day 21, 100 μl of β-Lg solution (10 mg / ml) was orally administered, and blood was collected after 1 hour and 2 weeks. On the other hand, β-Lg solution and Freund's complete adjuvant are mixed and emulsified, and injected into three subcutaneous sites (both dorsal side and buttocks) of a 3-month-old rabbit (white Japanese breed, male, Kitayama Labes). Thus, anti-β-Lg serum was obtained. The amount of β-Lg in the blood was measured by sandwich ELISA using a secondary antibody labeled with horseradish peroxidase (PO) using this antiserum as a primary antibody, and using blood one hour later. Further, anti-β-LgIgE in blood after 2 weeks was measured by ELISA using an anti-rat IgE antibody (manufactured by Nordic) labeled with β-Lg and PO. The results are shown in Table 3 below. As is apparent from the results shown in Table 3, the group in which sphingomyelin was administered at a dose of 0.1 mg / day or more per kg of body weight had a marked decrease in the mucosal permeability of β-Lg in the gastrointestinal tract compared to the control group. It was recognized and IgE production was suppressed, and thus it was found that sphingomyelin has an antiallergic action.

(Test Example 7)
The antioxidant effect of sphingomyelin was tested according to the method of “Test Example 1” in JP-A-11-209756.
Antioxidant activity of sphingomyelin was measured by the method of Osawa et al. (J. Agric. Food Chem., Vol. 35, pp. 809-812, 1987). That is, an equal amount of isotonic solution (10 mM phosphate buffer / 152 mM sodium chloride, pH 7.4) was mixed with rabbit blood, and centrifuged at 4 ° C., 1,500 × g (3,500 rpm) for 20 minutes. . This procedure is repeated three times, and an equal amount of hypotonic solution (10 mM phosphate buffer, pH 7.4) is mixed with the washed blood cells, and centrifuged at 4 ° C., 20,000 × g (11,000 rpm) for 40 minutes. did. And the antioxidant activity was investigated using the loose precipitation part (erythrocyte membrane ghost) obtained by repeating this operation 4 times. Using the sphingomyelin raw material of Example 1 of the present specification, sphingomyelin was prepared at each initial concentration (0 mM, 0.01 mM, 0.1 mM, 1 mM, 10 mM), and then mixed with an erythrocyte membrane ghost, Further, an oxidizing agent was added to carry out an oxidation reaction. Subsequently, after performing TBA reaction, the absorbance was measured at 532 nm to quantify the oxidized product. The antioxidant activity was calculated from the absorbance when each sphingomyelin was added with the absorbance when no sphingomyelin was added as 100%. In addition, it shows that oxidation of a erythrocyte membrane ghost is suppressed and antioxidant activity is so high that an light absorbency is low. The results are shown in Table 4. As is apparent from the results shown in Table 4, sphingomyelin was found to have a high antioxidant effect.

(Test Example 8)
The antioxidant effect of sphingomyelin was tested according to the method of “Test Example 2” in JP-A-11-209756.
Antioxidant activity of sphingomyelin was measured by the method of Nakayama et al. (Mutation Research, vol. 281, pp. 77-80, 1992). That is, Chinese hamster lung fibroblast V79 strain was seeded in a MEM medium containing 10% fetal calf serum (manufactured by Flow Laboratories) so that the number of cells was 200 per petri dish, and in the presence of 5% carbon dioxide, The cells were cultured at 37 ° C. for 5 days to obtain test cultured cells. Regarding the antioxidant activity of sphingomyelin, how much reduction in colony formation rate is recovered by adding sphingomyelin to cultured cells for testing, with the decrease in colony formation rate caused by hydrogen peroxide as an indicator of toxicity That's why it was judged.

  The above cultured cells for test are seeded on a plate, pre-cultured (cell adhesion) for 2 hours, and then each starting concentration (0 mM, 0.01 mM, 0 mM) using the sphingomyelin raw material of Example 1 of this specification. Sphingomyelin solution prepared to be 1 mM, 1 mM, 10 mM) was added and incubated for 4 hours to allow the cells to incorporate sphingomyelin prior to hydrogen peroxide. Next, hydrogen peroxide was added and allowed to react for 30 minutes to damage the cells. And after reaction, it culture | cultivated with the culture medium containing serum for 5 days. In addition, the density | concentration of hydrogen peroxide was set to 60 micromol which a colony formation rate falls to about several percent-40%. In addition, sphingomyelin was also examined in advance for its toxicity, and it was confirmed that the colony formation rate did not decrease due to its own toxicity. Antioxidant activity was evaluated after 5 days of culture, colony formation was confirmed, Giemsa staining was performed, the total number of colonies was counted, and cell viability when no sphingomyelin as a control was added but no hydrogen peroxide was added. The cell viability (%) with respect to 100%. In addition, it shows that the antioxidant activity of added sphingomyelin is so high that a cell viability is high. The results are shown in Table 5. As is apparent from the results shown in Table 5, sphingomyelin was found to have a high antioxidant effect.

(Test Example 9)
The sphingomyelin infection-protecting action was tested by the method described in JP-A-62-2208261.
Test for controlling incidence of diarrhea caused by pathogenic E. coli 30-day-old male SD rats were used as test animals, and the sphingomyelin raw material of Example 1 of the present specification was used for each test group consisting of 10 rats. Feeding myelin at 0 (control), 0.1, 1.0, 5.0, 10.0 mg / day and feeding each rat with a certain amount of pathogenic E. coli, causing diarrhea The rate was examined. The results are shown in Table 6. As can be seen from Table 6, it was found that the incidence of diarrhea was significantly reduced in rats administered with 1.0 mg or more of sphingomyelin per day.

(Test Example 10)
The anti-pathogenic E. coli O-157 infection-preventing action of sphingomyelin was tested according to the method of “Test Example 1” of JP-A-2001-2704.
・ Pathogenic Escherichia coli O-157 infection prevention test I
A 5-week-old BALB / c-type sterile mouse (20 mice) was orally ingested daily with saline (control group) and the sphingomyelin raw material (SPM group) of Example 1 herein. The intake of sphingomyelin was 5 mg / day. On the third day from the start of ingestion, pathogenic E. coli O-157 per mouse was orally administered at 8.5 × 10 6 cfu for infection. Even after infection, sphingomyelin was orally taken daily. Survival was observed for 8 days after administration of E. coli, and the survival rate of rats according to the number of days after administration of pathogenic E. coli O-157 is shown in Table 7. As is clear from the results in Table 7, the survival rate of sterile mice was increased by administration of sphingomyelin.

(Test Example 11)
The sphingomyelin was tested for its pathogenic E. coli O-157 infection-preventing action according to the method of “Test Example 2” of JP-A-2001-2704.
・ Pathogenic Escherichia coli O-157 Infection Prevention Test II
The survival rate of mice on the 8th day after administration of Escherichia coli was measured in the same manner as in Test Example 10 with the intake of sphingomyelin being 0.1 to 10 mg per day. The results are shown in Table 8. As is clear from the results shown in Table 8, the survival rate was remarkably improved in the group ingesting 1.0 mg / day or more of sphingomyelin.

(Test Example 12)
A 4-week-old hairless mouse (CD-1 (ICR) -nu / nu) was bred for 1 week and then bred for 3 weeks with the feed shown in Table 9. As a result, in a group (SPM group) administered with 40 mg / day of a milk-derived phospholipid fraction containing 15% sphingomyelin, hair grew at a rate of 7/10 animals. On the other hand, in the control group to which the milk-derived phospholipid fraction containing 15% sphingomyelin was not administered, hair grew only at a ratio of 1/10 animals.

(Test Example 13: Effect of sphingomyelin on EAE rats)
The effect of sphingomyelin on the treatment of demyelinating diseases was tested according to the method of “Example 3” of JP-A-2-250834.
The therapeutic effect of one type of demyelinating disease, EAE rat which is a multiple sclerosis model is shown.
A group of 5 Lewis rats (6 weeks old female), and a mixture of the same amount of brain homogenate from Freund's complete adjuvant (manufactured by Difco) as an EAE-induced antigen, converted to 80 mg brain homogenate. Rats were immunized with their hind footpads.
Sphingomyelin was administered intraperitoneally in the amount shown in Table 9 for 18 days from the day of immunization, and body weight measurement and EAE symptom observation were performed every day. EAE symptoms were rated on a 6-point scale. That is, 0: no abnormality, 1: tail paralysis, 2: hind limb weakness with tail paralysis, 3: hind limb paralysis with tail paralysis, 4: forelimb weakness with hind limb paralysis, 5: limb paralysis or front dying The effect was judged by the cumulative symptom degree.
Sphingomyelin is administered by suspending the sphingomyelin raw material of Example 1 in this specification in a sterilized 0.5% aqueous solution of sodium methylcellulose to a concentration of 1 mg / ml or 2 mg / ml and administering it intraperitoneally. did. Only saline was administered as a control. The results are shown in Table 10 below. As is apparent from the results in Table 10, sphingomyelin significantly suppressed the onset of EAE compared to the control using only physiological saline. As a result, it was found that it can be used effectively for the treatment and prevention of multiple sclerosis.

(Test Example 14: Sphingomyelin antibody production inhibitory effect)
The effect of sphingomyelin on the treatment of demyelinating diseases was tested according to the method of “Example 4” of JP-A-2-250834.
The sphingomyelin EAE onset prevention mechanism was examined. That is, 0.2 ml of 50% sheep erythrocytes (SRBC) was intraperitoneally administered on the 14th day to rats administered with the physiological saline and sphingomyelin 2 mg / kg shown in Test Example 13, and on the 18th day, The spleen was removed from the rat and the single cell suspension was aseptically adjusted. Red blood cells were removed by hemolysis, and then washed with RPMI-1640 medium to prepare a cell suspension with a cell density of 2 × 10 9 cells / ml.
Plaque-forming cells against sheep erythrocytes by the method of Jerne were counted and used as IgMPFC numbers. The results are shown in Table 11. From the results shown in Table 11, it is confirmed that sphingomyelin has an antibody production inhibitory activity, and it is considered that EAE onset is inhibited based on the activity.

(Test Example 15)
The anti-pigmenting action of sphingomyelin was tested according to the method described in JP-A-1-163112.
Using 8MOP-treated phototoxic pigmented Wester Maple GP, a substance that promotes pigmentation, 50 μl of sample was cut once a day on the shaved back of ICR female mice (6 weeks old, 5 mice per group). It was applied to a range of 4 cm 2 for 8 weeks, and the degree of pigmentation shown in Table 12 as anti-pigmentation effects and side effects was evaluated by a 4-point evaluation method (+ indicates decolorization effect, − indicates side effects). The sample was used by dissolving the sphingomyelin raw material of Example 1 of this specification to 5%. Moreover, the thing which does not apply | coat anything was made into the control | contrast. The results are shown in Table 13. As is clear from the results in Table 13, it was found that sphingomyelin has no side effects and is excellent in anti-pigment effect.

(Test Example 16)
The anti-pigmentation effect by oral administration of sphingomyelin was tested.
A-1 female guinea pig (weight approximately 400 g) is removed from the back, and the back is irradiated with ultraviolet rays (UVA (max. 360 nm) 30.3 kJ / m ² , UVB (max. 312 nm) 4.8 kJ / m ² ). The test was performed once a day for 4 days. Thereafter, a group (group A) in which 10 g / kg of guinea pig body weight is administered without administration of sphingomyelin, and a group (group B) in which 2 mg / kg of guinea pig body weight is administered using the sphingomyelin raw material of Example 1 as sphingomyelin. A group (group C) in which 5 mg / kg of guinea pig body weight is administered with the sphingomyelin raw material of Example 1 as sphingomyelin, and a group (group D) in which 10 mg / kg of guinea pig body weight is administered with the sphingomyelin raw material of Example 1 as sphingomyelin. Were divided into 4 test groups (10 animals in each group). Each was orally administered once a day with a sonde and reared for 4 weeks. The sphingomyelin raw material of Example 1 was suspended in 10 g of physiological saline and orally administered to groups B to D, respectively. At the start of sample administration and at the end of sample administration, the effect on the pigmentation of the guinea pig dorsal skin was measured with a MINOLTA color difference meter (CHROMA METER CR-200), and the brightness recovery rate from the start of sample administration was calculated. did. The results are shown in Table 14.

From the results shown in Table 14, the brightness recovery rate after oral administration for 4 weeks was as low as 31% in Group A, but 48% in Group B, 62% in Group C, and 78% in Group D. It rose up to 2.5 times compared to the group.
From this, it was found that the lightness recovery rate was increased by oral administration of sphingomyelin. That is, it was confirmed that oral administration of sphingomyelin has an anti-pigmentation effect. As for the dose, it was found that the effect was observed when 2 mg or more of sphingomyelin was orally administered per kg of guinea pig body weight, and the effect was remarkable when 5 mg or more was orally administered.

(Test Example 17)
The anti-inflammatory action of sphingomyelin was tested by the method described in JP-A-1-163125.
The anti-inflammatory effect of sphingomyelin was tested by the carrageenan footpad edema method.
That is, according to the method of Winter et al. (Proceedings of the Society for Experimental Biology & Medicine, Vol. 111, pp. 554, 1962), a Wistar male rat (weight, 110-130 g, 8 per group) was added to 0.5% carboxymethylcellulose aqueous solution. The suspended test substance shown in Table 15 was orally administered (100 mg / kg). One hour later, 0.1 ml of 1% λ-carrageenan / physiological saline solution as a pro-inflammatory substance was subcutaneously administered to the rat's hindlimb footpad to induce edema. Each footpad volume was measured before and after the administration of the inflammatory substance, and the increase rate (V1) of the footpad dose was determined. As a control group, the rate of increase in paw volume (V0) when λ-carrageenin was similarly injected into rats administered with a 0.5% carboxymethylcellulose aqueous solution containing no test substance was measured, and (V0-V1) × The carrageenin edema suppression rate (%) was calculated by the calculation formula of 100 / V0, and was defined as the anti-inflammatory activity of the test substance. It shows that anti-inflammatory activity is so high that this value is large. Table 15 shows the measured values 5 hours after λ-carrageenin injection. As is clear from the results shown in Table 15, sphingomyelin showed an edema suppression rate stronger than indomethacin and sialic acid, and was found to have a strong anti-inflammatory effect.

(Example 2)
After mixing the raw materials with the formulation shown in Table 16, it was molded into 1 g by a conventional method and tableted to produce the medicament of the present invention as a tablet.

Example 3
50 g of sphingomyelin raw material (phospholipid 700, manufactured by Fonterra) having a sphingomyelin content of 25% is dissolved in 4950 g of deionized water as an agent of the present invention, heated to 50 ° C., and then TK homomixer (TK ROBO MICS; specialized machine) Manufactured by Kogyo Co., Ltd.) and stirred and mixed at 6000 rpm for 30 minutes to obtain a sphingomyelin solution having a sphingomyelin content of 250 mg / 100 g. 4.0 kg of this sphingomyelin solution, 5.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 18.0 kg of dextrin, 6.0 kg of mineral mixture, 1.95 kg of vitamin mixture, emulsifier 2 0.0 kg, stabilizer 4.0 kg, and fragrance 0.05 kg were mixed, filled in a 200 ml retort pouch, and retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Manufacturing Co., Ltd.) at 121 ° C. And sterilized for 20 minutes to produce 50 kg of a liquid nutritional composition containing the agent of the present invention. This liquid nutritional composition contained 20 mg of sphingomyelin per 100 g.

Example 4
10 g of sphingomyelin raw material having a sphingomyelin content of 10% (phospholipid 500, manufactured by Fonterra) was dissolved in 700 g of deionized water as an agent of the present invention, heated to 50 ° C., and then treated with an ultradisperser (ULTRA-TURRAX T-25). And manufactured by IKA Japan) at 9500 rpm for 30 minutes. To this solution, 40 g of sorbitol, 2 g of acidulant, 2 g of fragrance, 5 g of pectin, 5 g of whey protein concentrate, 1 g of calcium lactate and 235 g of deionized water were added and mixed with stirring. After sterilizing at 20 ° C. for 20 minutes, the bottle was sealed and 5 gel bags (200 g) containing the agent of the present invention were prepared. The gel-like foods thus obtained were not found to have any precipitation or the like, and no abnormal flavor was felt. The gel food contained 100 mg of sphingomyelin per 100 g.

(Example 5)
After dissolving 2 g of acidulant in 700 g of deionized water, 10 g of sphingomyelin raw material having a sphingomyelin content of 25% (phospholipid 700, manufactured by Fonterra) was dissolved as an agent of the present invention, heated to 50 ° C., Ultradis The mixture was stirred and mixed at 9500 rpm for 30 minutes with a parser (ULTRA-TURRAX T-25; manufactured by IKA Japan). After adding 100 g of maltitol, 20 g of reduced starch syrup, 2 g of fragrance and 166 g of deionized water, it is filled into a 100 ml glass bottle, sterilized at 90 ° C. for 15 minutes, sealed, and 10 drinks (100 ml) containing the agent of the present invention Prepared). In all of the beverages obtained in this way, no precipitation was observed and no abnormal flavor was felt. In addition, this drink contained 250 mg of sphingomyelin per 100 g.

(Example 6)
2 kg of sphingomyelin raw material (SM-4, manufactured by Corman) having a sphingomyelin content of 4% was dissolved in 98 kg of deionized water as an agent of the present invention, heated to 50 ° C., and then TK homomixer (MARK II 160 type; special machine) (Manufactured by Kogyo Co., Ltd.) and stirring and mixing at 3600 rpm for 40 minutes to obtain a sphingomyelin solution having a sphingomyelin content of 80 mg / 100 g. 10 kg of this sphingomyelin solution, 12 kg of soybean meal, 14 kg of nonfat dry milk, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, 2 kg of mineral mixture Was sterilized at 120 ° C. for 4 minutes to produce 100 kg of dog breeding feed formulated with the agent of the present invention. The dog feed contained 8 mg of sphingomyelin per 100 g.

  The medicament which is a preventive or therapeutic agent for various diseases comprising the sphingomyelin of the present invention as an active ingredient, or a food or drink or feed containing these agents should be used for the prevention or treatment of each disease, improvement of symptoms, etc. Can be very useful.

Claims (2)

A pharmaceutical composition for preventing sickness, comprising sphingomyelin as an active ingredient. 2. The pharmaceutical composition for preventing sickness according to claim 1, wherein sphingomyelin is derived from milk.

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